The role of IFNγ in higher brain function: in health and under chronic stress

Tese de Doutoramento em Ciências da SaúdeThe neuroimmunology field is at an exciting stage due to a set of revolutionary discoveries challenging the now old-fashioned dogma of the brain being “protected” from the peripheral immune system action. Immune components such as T lymphocytes and the cytokines they produce, once regarded as detrimental to the brain, are now considered integrant parts of the healthy nervous system since their regulated actions control immune surveillance but also modulate higher brain functions. The cytokine interferon-gamma (IFNγ), produced mainly by T lymphocytes, is a potent pro-inflammatory molecule, whose levels are altered in many neuropsychiatric and neurodegenerative diseases. Though studies assessing the effects of this cytokine, when administered into the brain, have shown that it affects different cellular and synaptic mechanisms underlying behavioural dimensions, it is still unclear whether this is a collateral damage of the inflammatory response or if IFNγ indeed plays a role in the modulation of non-pathological brain function. As so, we sought to explore the role of this cytokine in the modulation of brain function in physiological conditions and also after exposure to chronic stress – a paradigm known to trigger the development of psychiatric complications and also accelerate neurodegenerative processes. In the first part of the thesis (2nd Chapter) we demonstrate that, in a healthy brain, the absence of IFNγ enhances dorsal hippocampus plasticity and associated cognitive function. At the structural level, an enlargement of the dorsal hippocampus volume contrasted with the absence of alterations observed in the ventral part, highlighting that the effects of this cytokine are more selective for cognitive behaviours. Moreover, the absence of this cytokine amplifies neuroplastic phenomena in the dorsal hippocampus, namely neurogenesis, size of neuronal dendritic arborisations and presynaptic functioning, most likely contributing for the enhanced cognitive performance. On the 3rd chapter, we demonstrate that there are gender-differences on the behavioural phenotype of IFNγ KO mice, and discuss the possible association of estrogen and the IFNγ expression in the central nervous system. In the following chapter (4th Chapter) we describe the optimisation of a chronic unpredictable stress (CUS) paradigm for use in C57BL/6 mice, a strain with higher resistance to stress. This mice model of stress-related disorders exhibits, beyond the stress-related neuroendocrine and behavioural alterations, mild changes in thymic cellular populations and relevant splenic myeloid cellular alterations, with an increased number of neutrophils as the most striking change. At last (5th Chapter), we discuss the contributory role of IFNγ for the development of the immune maladaptive response to chronic stress. By submitting mice to the optimized CUS protocol, it was observed that mRNA levels of Ifnγ are elevated in the brain, specifically in the medial prefrontal and orbitofrontal cortices. Moreover, exposure to chronic stress leads to an increase of the adrenergic innervation of the spleen as to alterations on the percentage of neutrophils and monocytes/macrophages populations in the spleen. Importantly, the absence of this cytokine blunts the stress-related changes on these cell populations in the spleen. The recognition of the proinflammatory cytokine – IFNγ, as a negative regulator of hippocampal plasticity and associated cognitive function, together with its contributory role for the stress-related immune dysfunction, suggests that this cytokine may articulate the complex network that underlies the inflammatory component of neuropsychiatric disorders.A área da neuroimunologia está a atravessar uma fase excitante devido a um conjunto de descobertas revolucionárias que desafiam o seu agora antiquado dogma que visiona o cérebro como um órgão “protegido” da ação do sistema imunitário periférico. Componentes imunitários, tal como os linfócitos T e respetivas citocinas que estes produzem, outrora vistos apenas como prejudiciais para o cérebro, são agora considerados partes integrantes do sistema nervoso, uma vez que a sua ação regulada controla a vigilância imunitária mas também a modulação de funções cerebrais superiores. O interferão gama (IFNγ), uma citocina produzida principalmente por linfócitos T, é uma molécula proinflamatória cujos níveis estão alterados em diversas doenças neuropsiquiátricas e neurodegenerativas. Apesar de estudos demonstrarem que a administração desta citocina no cérebro afeta diferentes mecanismos celulares e sinápticos que estão na base de dimensões comportamentais, não é ainda claro se este efeito é um dano colateral da resposta inflamatória ou se o IFNγ tem de fato um papel na modulação da função do cérebro num contexto não patológico. Como tal, iremos aqui explorar o papel desta citocina na modulação de funções cerebrais em condições fisiológicas como também após exposição ao stress crónico – um paradigma que desencadeia o desenvolvimento de complicações psiquiátricas e acelera processos neurodegenerativos. Na primeira parte da presente tese (2º Capítulo) nós demonstramos que a ausência de IFNγ no cérebro saudável leva a uma melhoria da plasticidade do hipocampo dorsal e função cognitiva associada. Ao nível estrutural, um aumento do volume do hipocampo dorsal contrasta com a ausência de alterações volumétricas na parte ventral, sublinhando assim que esta citocina afeta seletivamente o comportamento cognitivo. A ausência desta citocina leva também a uma amplificação dos fenómenos neuroplásticos do hipocampo dorsal, nomeadamente da neurogénese, o tamanho da arborização dendrítica neuronal e o funcionamento pré-sináptico, contribuindo, muito provavelmente, para a melhoria da performance cognitiva. No 3º capítulo, nós demonstramos que existem diferenças entre géneros no fenótipo comportamental dos murganhos IFNγ KO, e discutimos a possível associação entre o estrogénio e a expressão de IFNγ no sistema nervoso central. No capítulo seguinte (4º Capítulo) descrevemos a otimização de um paradigma de stress crónico imprevisível (CUS) para murganhos C57BL/6, uma estirpe que apresenta uma maior resistência ao stress. Este modelo de murganho de disfunção associada ao stress apresenta para além das alterações neuroendócrinas e comportamentais relacionadas com o stress, alterações moderadas nas populações celulares do timo e importantes alterações celulares mielóides no baço, sendo o aumento de neutrófilos a alteração mais impressionante. Por último (5º Capítulo), discutimos o papel contributório desta citocina para o desenvolvimento da resposta imunitária maladaptativa ao stress crónico. Após expor murganhos ao protocolo otimizado de CUS foi observado um aumento nos níveis de mRNA de Ifnγ, mais especificamente nos córtices préfrontal medial e orbitofrontal. Para além disso, a exposição ao stress crónico leva a um aumento da inervação adrenérgica do baço assim como a alterações nas percentagens das populações de neutrófilos e monócitos/macrófagos no baço. A ausência de IFNγ preveniu as alterações induzidas pelo stress crónico nestas populações celulares do baço. O reconhecimento da citocina pró-inflamatória – IFNγ, como um regulador negativo da plasticidade hippocampal e função cognitiva associada, juntamente com o seu papel contributório para a disfunção imunitária associada com o stress sugere que esta citocina poderá articular a rede complexa que está na base da componente inflamatória das doenças neuropsiquiátricas.This work was funded by the European Commission (FP7): “SwitchBox” (Contract HEALTH-F2- 2010-259772) and co-financed by the Portuguese North Regional Operational Program (ON.2 – O Novo Norte) under the National Strategic Reference Framework (QREN), through the European Regional Development Fund (FEDER). Susana Isabel Gonçalves Monteiro was supported by a PhD fellowship from the Portuguese Science Foundation (FCT)/MEC with the reference SFRH/BD/69311/2010

Use of immunohistochemical techniques to investigate museum specimens of neurological disease

Mestrado em Métodos BiomolecularesOs espécimes de museu são um recurso valioso tanto para o ensino como para a investigação, mas geralmente têm sido utilizados para demonstrar a morfologia macroscópica. O objectivo da presente dissertação é determinar se modernas técnicas neuropatológicas, tal como a histologia e a imunohistoquímica, podem ser aplicadas com sucesso a espécimes de museu preservados por períodos superiores a 50 anos. Poderá ser esperado que a qualidade do material histológico recuperado de espécimes de museu seja afectada por vários factores. Estes incluem a idade do espécime, o tipo e a duração da fixação/preservação, condições do acondicionamento do espécime, informação relativa ao paciente e o período post-mortem. É importante compreender como é que os métodos de preservação mudaram com o passar dos tempos, consequentemente uma breve revisão desta história foi feita. É descrito o processo de selecção dos espécimes para este estudo e é apresentada uma investigação às características das diferentes soluções de montagem. Grande parte desta dissertação refere-se ao desenvolvimento de formas que permitam a aplicação de técnicas histológicas modernas a espécimes de museu. É demonstrado que uma pós-fixação em formaldeído 10% e um ajuste do pH da solução corante de eosina para um intervalo entre 4.8 e 5.0, são especialmente importantes em tecidos envelhecidos. Foi obtida uma coloração imunohistoquímica satisfactória utilizando o sistema EnVision+ (Dako), um polímero acoplado com HRP, com o anticorpo anti-neurofilamento (Dako). No entanto, apenas alcançou-se resultados satisfatórios com cortes de crióstato, os quais, aparentemente, produzem melhor coloração imunohistoquímica quando comparados com cortes de tecidos fixados em formaldeído e incluídos em parafina (FFPE), sugerindo assim, que o processamento em solventes orgânicos e as altas temperaturas da parafina derretida alteram a conformação do antigénio impedindo a detecção imunohistoquímica. Um estudo detalhado de três casos datados de 1953, 1954 e 1955 confirma que técnicas modernas (incluíndo imunohistoquímica) podem ser utilizadas em tecidos envelhecidos a um ponto de serem consideradas diagnosticamente válidas. Este estudo demonstra que com ajustes cuidados aos protocolos é possível obter-se notáveis resultados histológicos de alta qualidade em tecidos que foram preservados por muitos anos. Confirma também, que espécimes de museu representam um valioso recurso para ensino e para a investigação a um nível ultraestrutural. ABSTRACT: Museum Specimens are a valuable resource both for teaching and research but have generally been used to show the gross morphology. The aim of this dissertation is to determine if modern neuropathological techniques, such as histology and immunohistochemistry, can be applied successfully in museum specimens that have been preserved for periods in excess of 50 years. It may be expected that many factors could affect the quality of histological material retrieved from museum specimens. These include the age of the specimen, type and length of fixation/preservation, storage conditions, patient information and post-mortem period. It is important to understand how preservation methods had changed over time, and therefore, the complex history of “potting” is reviewed. The process of selecting specimens for this study is described and an investigation into the characteristics of mounting solutions is presented. The major part of the dissertation concerns the development of ways in which to apply modern histological techniques to museum specimens. It is shown that post-fixation in formaldehyde 10% and an adjustment of the eosin stain solution to a pH ranging between 4.8 and 5.0 are especially important in older tissues. Satisfactory immunohistochemistry staining was obtained using EnVision + (Dako) system, a HRP labeled polymer, with anti-neurofilament antibody (Dako). Nevertheless, successful results were only achieved with cryostat sections, as they appeared to produce better immunohistochemistry staining when compared to formalin-fixed paraffin-embedded (FFPE) sections, suggesting that processing in organic solvents and high temperatures of molten paraffin alters the conformation of the antigen hampering immunohistochemistry detection. A detailed study of three cases from 1953, 1954 and 1955 confirms that modern techniques (including immunohistochemistry) can be used in aged tissue to the point where they are useful diagnostically. This study shows that with careful adjustment to protocols it is possible to achieve remarkably high quality histological results in tissues that have been preserved for many years. It confirms that specimens in museums represent a valuable resource for teaching and research at an ultrastructural level

Pathophysiology and therapeutic approaches for spinal cord injury

Spinal cord injury (SCI) is a disabling condition that disrupts motor, sensory, and autonomic functions. Despite extensive research in the last decades, SCI continues to be a global health priority affecting thousands of individuals every year. The lack of effective therapeutic strategies for patients with SCI reflects its complex pathophysiology that leads to the point of no return in its function repair and regeneration capacity. Recently, however, several studies started to uncover the intricate network of mechanisms involved in SCI leading to the development of new therapeutic approaches. In this work, we present a detailed description of the physiology and anatomy of the spinal cord and the pathophysiology of SCI. Additionally, we provide an overview of different molecular strategies that demonstrate promising potential in the modulation of the secondary injury events that promote neuroprotection or neuroregeneration. We also briefly discuss other emerging therapies, including cell-based therapies, biomaterials, and epidural electric stimulation. A successful therapy might target different pathologic events to control the progression of secondary damage of SCI and promote regeneration leading to functional recovery.This work has been funded by National funds, through the Foundation for Science and Technology (FCT)-project UIDB/50026/2020, UIDP/50026/2020 and project EXPL/MED-PAT/0931/2021. Financial support was also provided from Prémios Santa Casa Neurociências-Prize Melo e Castro for Spinal Cord Injury Research (MC-18-2021)

The central nervous system source modulates microglia function and morphology in vitro

The regional heterogeneity of microglia was first described a century ago by Pio del Rio Hortega. Currently, new information on microglia heterogeneity throughout central nervous system (CNS) regions is being revealed by high-throughput techniques. It remains unclear whether these spatial specificities translate into different microglial behaviors in vitro. We cultured microglia isolated from the cortex and spinal cord and analyzed the effect of the CNS spatial source on behavior in vitro by applying the same experimental protocol and culture conditions. We analyzed the microglial cell numbers, function, and morphology and found a distinctive in vitro phenotype. We found that microglia were present in higher numbers in the spinal-cord-derived glial cultures, presenting different expressions of inflammatory genes and a lower phagocytosis rate under basal conditions or after activation with LPS and IFN-γ. Morphologically, the cortical microglial cells were more complex and presented longer ramifications, which were also observed in vivo in CX3CR1+/GFP transgenic reporter mice. Collectively, our data demonstrated that microglial behavior in vitro is defined according to specific spatial characteristics acquired by the tissue. Thus, our study highlights the importance of microglia as a source of CNS for in vitro studies.This work was funded by the Santa Casa Neuroscience Awards—Prize Melo e Castro for Spinal Cord Injury Research (MC-18-2021), and it was partially funded by the Wings for Life Spinal Cord Research Foundation (WFL-ES-03/19). This work was also funded by national funds through the Foundation for Science and Technology (FCT)—projects UIDB/50026/2020, UIDP/50026/2020, and EXPL/MED-PAT/0931/2021, and by the project NORTE-01-0145-FEDER-000039, supported by the Norte Portugal Regional Operational Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (ERDF). We would like to acknowledge the support given by the Portuguese Foundation of Science and Technology to AGP (2020.07534.BD), AM (UMINHO/BIL-CNCG/2022/16), SM (CEECIND/01902/2017), and NAS (CEECIND/04794/2007)

Preclinical assessment of mesenchymal-stem-cell-based therapies in spinocerebellar ataxia type 3

The low regeneration potential of the central nervous system (CNS) represents a challenge for the development of new therapeutic strategies for neurodegenerative diseases, including spinocerebellar ataxias. Spinocerebellar ataxia type 3 (SCA3)—or Machado–Joseph disease (MJD)—is the most common dominant ataxia, being mainly characterized by motor deficits; however, SCA3/MJD has a complex and heterogeneous pathophysiology, involving many CNS brain regions, contributing to the lack of effective therapies. Mesenchymal stem cells (MSCs) have been proposed as a potential therapeutic tool for CNS disorders. Beyond their differentiation potential, MSCs secrete a broad range of neuroregulatory factors that can promote relevant neuroprotective and immunomodulatory actions in different pathophysiological contexts. The objective of this work was to study the effects of (1) human MSC transplantation and (2) human MSC secretome (CM) administration on disease progression in vivo, using the CMVMJD135 mouse model of SCA3/MJD. Our results showed that a single CM administration was more beneficial than MSC transplantation—particularly in the cerebellum and basal ganglia—while no motor improvement was observed when these cell-based therapeutic approaches were applied in the spinal cord. However, the effects observed were mild and transient, suggesting that continuous or repeated administration would be needed, which should be further tested.This research was funded by the National Ataxia Foundation (NAF) and by Portuguese national funds, through the Foundation for Science and Technology (FCT)—projects UIDB/50026/2020, UIDP/50026/2020, POCI-01-0145-FEDER-029206, and through the Santa Casa Neuroscience Awards (Santa Casa da Misericórdia Lisboa)—project MC-04/17. Additionally, this project was funded by the ICVS Scientific Microscopy Platform, a member of the national infrastructure PPBI—Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122). S.C.S. received an individual fellowship within the project TUBITAK/0007/2014. The FCT funded individual fellowships to J.S C., A.N.-C., B.M.- P., F.G.T., R.L., S.M., N.A.S., C.S.-C., and S.D.-S. (SFRH/BD/140624/2018, SFRH/BPD/118779/2016, SFRH/BD/120124/2016, SFRH/BPD/118408/2016, PD/BDE/127836/2016, CEECIND/01902/2017, CEECIND/04794/2017, CEECIND/03887/2017, and CEECIND/00685/2020)

A Lei da Mediação de Conflitos: estudos sobre a sua aplicação

Financiamento de MEDLaw - FCT UIDB/04112/2020.Os dez anos de vigência da Lei da Mediação em Portugal constituíram o mote para a compilação nesta obra de diversos estudos empírico-dogmáticos sobre a sua aplicação, analisando-se questões prementes como a voluntariedade ou obrigatoriedade da mediação, a executoriedade do acordo de mediação e a Convenção de Singapura, as exigências processuais e a suspensão dos prazos de prescrição e caducidade com o recurso à mediação, a organização associativa dos mediadores e a importância da sua formação, o funcionamento dos sistemas públicos de mediação, bem como novas áreas de aplicação da mediação, em especial no domínio administrativo e na recuperação extrajudicial de empresas, e ainda a relevância do desenvolvimento científico sobre este meio de resolução de conflitos. Ao regulamentar num único diploma, pela primeira vez no nosso ordenamento jurídico, a mediação pública e privada, a Lei n.º 29/2013, de 19 de abril, constituiu um marco legislativo. Dez anos volvidos, importava refletir sobre a sua aplicação prático-jurídica, norteados pelo objetivo de promover o estudo e a efetiva implementação da mediação de conflitos em Portugal. Esta obra constitui o output desenvolvido no âmbito do projeto de investigação MEDLAW, com o apoio da Fundação para a Ciência e Tecnologia, no âmbito do financiamento base atribuído ao polo de Leiria do Instituto Jurídico Portucalense, com a ref. UIDB/04112/2020.info:eu-repo/semantics/publishedVersio

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Immunocompromised patients with acute respiratory distress syndrome: Secondary analysis of the LUNG SAFE database

Background: The aim of this study was to describe data on epidemiology, ventilatory management, and outcome of acute respiratory distress syndrome (ARDS) in immunocompromised patients. Methods: We performed a post hoc analysis on the cohort of immunocompromised patients enrolled in the Large Observational Study to Understand the Global Impact of Severe Acute Respiratory Failure (LUNG SAFE) study. The LUNG SAFE study was an international, prospective study including hypoxemic patients in 459 ICUs from 50 countries across 5 continents. Results: Of 2813 patients with ARDS, 584 (20.8%) were immunocompromised, 38.9% of whom had an unspecified cause. Pneumonia, nonpulmonary sepsis, and noncardiogenic shock were their most common risk factors for ARDS. Hospital mortality was higher in immunocompromised than in immunocompetent patients (52.4% vs 36.2%; p &lt; 0.0001), despite similar severity of ARDS. Decisions regarding limiting life-sustaining measures were significantly more frequent in immunocompromised patients (27.1% vs 18.6%; p &lt; 0.0001). Use of noninvasive ventilation (NIV) as first-line treatment was higher in immunocompromised patients (20.9% vs 15.9%; p = 0.0048), and immunodeficiency remained independently associated with the use of NIV after adjustment for confounders. Forty-eight percent of the patients treated with NIV were intubated, and their mortality was not different from that of the patients invasively ventilated ab initio. Conclusions: Immunosuppression is frequent in patients with ARDS, and infections are the main risk factors for ARDS in these immunocompromised patients. Their management differs from that of immunocompetent patients, particularly the greater use of NIV as first-line ventilation strategy. Compared with immunocompetent subjects, they have higher mortality regardless of ARDS severity as well as a higher frequency of limitation of life-sustaining measures. Nonetheless, nearly half of these patients survive to hospital discharge. Trial registration: ClinicalTrials.gov, NCT02010073. Registered on 12 December 2013

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved